The objective of this research is to undertake a detailed analysis of an under-investigated class of proteins: the mammalian phosphatidylinositol/phosphatidylcholine transfer proteins (PITPs). The functions and mechanisms of function of PITPs in mammalian cells remain to be elucidated. The research plan is designed to identify mechanisms of function of a specific mammalian PITP isoform (PITPalpha). This proposal is founded on our creation and characterization of a PITPalpha knockout mouse. PITP-/- mice suffer from spinocerebellar degeneration, hypoglycemia, and failure to absorb dietary fat and fat-soluble vitamins across the small intestine (i.e. a chylomicron retention disease). These phenotypes manifest themselves upon birth and PITPalpha-/- mice rapidly succumb to these disorders. The PITPalpha-/- mouse is an ideal disease model in that it is born alive, but manifests powerful phenotypes. The rates of phenotype onset are rapid. Using this unique model, we will undertake three lines of investigation. First, we will restore PITPalpha expression to the small intestine of the PITPalpha-/- mouse to test our hypothesis that the dietary fat malabsorption syndrome is a primary factor in hypoglycemia and spinocerebellar degeneration. In this effort, we propose to develop a cultured enterocyte model in which chylomicron assembly and transport can be studied in detail. Second, our data indicate the PITPalpha-deficient neurons exhibit intrinsic defects; most likely in signaling through the so-called 'survival pathways'. Using genetic, biochemical and signaling approaches we will test this hypothesis in the PITPalpha-/- model. These studies will be complemented by exploitation of a novel Drosophila system for study of PITPct signaling functions. Third, we will use genome engineering approaches to functionally dissect the physiological functions of individual PITPalpha phospholipid transfer activities. In particular, we will test whether the key physiological function of PITPalpha is to facilitate phosphoinositide synthesis and, if so, what specific phosphoinositides are regulated in a PITPalpha-dependent manner and what signaling pathways are involved. PITPs play central roles in regulating signal transduction pathways that interface with diverse cellular processes. We now establish that these functional interfaces are relevant to human chylomicron retention disease, disorders of glucose homeostasis, and spinocerebellar degeneration. The Bankaitis laboratory is uniquely poised to address questions of mechanism of PITPalpha function as it has developed unique experimental systems for analysis.